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CSN-mediated deneddylation differentially modulates Ci(155) proteolysis to promote Hedgehog signalling responses.

Wu JT, Lin WH, Chen WY, Huang YC, Tang CY, Ho MS, Pi H, Chien CT - Nat Commun (2011)

Bottom Line: Here, we show that in COP9 signalosome (CSN) mutants, in which deneddylation of SCF(Slimb) is inactivated, Ci is destabilized in low-to-intermediate Hh signalling cells.The status of Ci phosphorylation and the level of E1 ubiquitin-activating enzyme are tightly coupled to this CSN regulation.We propose that the affinity of substrate-E3 interaction, ligase activity and E1 activity are three major determinants for substrate ubiquitylation and thereby substrate degradation in vivo.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Medicine, College of Medicine, National Taiwan University, Taipei 100, Taiwan.

ABSTRACT
The Hedgehog (Hh) morphogen directs distinct cell responses according to its distinct signalling levels. Hh signalling stabilizes transcription factor cubitus interruptus (Ci) by prohibiting SCF(Slimb)-dependent ubiquitylation and proteolysis of Ci. How graded Hh signalling confers differential SCF(Slimb)-mediated Ci proteolysis in responding cells remains unclear. Here, we show that in COP9 signalosome (CSN) mutants, in which deneddylation of SCF(Slimb) is inactivated, Ci is destabilized in low-to-intermediate Hh signalling cells. As a consequence, expression of the low-threshold Hh target gene dpp is disrupted, highlighting the critical role of CSN deneddylation on low-to-intermediate Hh signalling response. The status of Ci phosphorylation and the level of E1 ubiquitin-activating enzyme are tightly coupled to this CSN regulation. We propose that the affinity of substrate-E3 interaction, ligase activity and E1 activity are three major determinants for substrate ubiquitylation and thereby substrate degradation in vivo.

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Models for SCFSlimb-mediated Ci155 proteolysis.Successful substrate polyubiquitylation requires collaboration of three components: adequate substrate–enzyme interaction time, persistent neddylated E3 ligase activity and high levels of activated ubiquitin supply. Green dot, ubiquitin; blue dot, Nedd8; and red dot, phosphate group. (a) For fully phosphorylated Ci155 that binds strongly to SCF in no-to-low Hh signalling regions, long substrate–enzyme binding duration allows complete polyubiquitylation, despite temporary inactivation of SCF ligase by CSN-mediated deneddylation. (b) In low-to-intermediate Hh signalling regions as well as in the presence of DN-GSK3β, partially phosphorylated Ci155 associates with SCF weakly. Dissociation of Ci155 from the SCFSlimb complex following CSN-mediated deneddylation of SCF disrupts processive ubiquitylation of Ci155. (c) Limited supply of activated ubiquitin could disrupt processive ubiquitylation of fully phosphorylated Ci155 in no-to-low Hh regions in the presence of CSN deneddylation. (d) CSN mutations allow the processivity of polyubiquitylation mediated by constantly neddylated SCF, which compensates for the short duration of interaction between SCFSlimb and Ci155 in low-to-intermediate Hh signalling regions or in the presence of DN-GSK3β. (e) In no-to-low Hh regions, limited supply of activated ubiquitin in uba1-dsRNA cells interrupts Ci155 polyubiquitylation, which is suppressed by constitutively neddylated SCF in CSN mutants.
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f7: Models for SCFSlimb-mediated Ci155 proteolysis.Successful substrate polyubiquitylation requires collaboration of three components: adequate substrate–enzyme interaction time, persistent neddylated E3 ligase activity and high levels of activated ubiquitin supply. Green dot, ubiquitin; blue dot, Nedd8; and red dot, phosphate group. (a) For fully phosphorylated Ci155 that binds strongly to SCF in no-to-low Hh signalling regions, long substrate–enzyme binding duration allows complete polyubiquitylation, despite temporary inactivation of SCF ligase by CSN-mediated deneddylation. (b) In low-to-intermediate Hh signalling regions as well as in the presence of DN-GSK3β, partially phosphorylated Ci155 associates with SCF weakly. Dissociation of Ci155 from the SCFSlimb complex following CSN-mediated deneddylation of SCF disrupts processive ubiquitylation of Ci155. (c) Limited supply of activated ubiquitin could disrupt processive ubiquitylation of fully phosphorylated Ci155 in no-to-low Hh regions in the presence of CSN deneddylation. (d) CSN mutations allow the processivity of polyubiquitylation mediated by constantly neddylated SCF, which compensates for the short duration of interaction between SCFSlimb and Ci155 in low-to-intermediate Hh signalling regions or in the presence of DN-GSK3β. (e) In no-to-low Hh regions, limited supply of activated ubiquitin in uba1-dsRNA cells interrupts Ci155 polyubiquitylation, which is suppressed by constitutively neddylated SCF in CSN mutants.

Mentions: In this study, we show that CSN-mediated deneddylation of SCFSlimb is critical for the Ci155 stability in low-to-intermediate Hh regions in which partially phosphorylated Ci155 has a reduced affinity for the ubiquitylation machinery SCFSlimb. The effect of CSN deneddylation on SCFSlimb likely reduces ubiquitylation processivity, thus preserving 'conditionally stable Ci155' in these low-to-intermediate Hh regions. We propose a broader view for CRL substrate degradation in vivo, determined by three interdependent factors: substrate-enzyme affinity, deneddylation-regulated CRL activity and the supply of Uba1-activated Ub. We propose that steady-state Ci155 levels are low in the low Hh signalling regions because processive polyubiquitylation of the tightly associated SCFSlimb-Ci155 complex proceeds even with intermittent SCFSlimb inactivation by CSN-mediated deneddylation (Fig. 7a). In low-to-intermediate Hh regions, the steady-state Ci155 levels are sensitive to CSN-mediated deneddylation as a result of weakened SCFSlimb-Ci155 association, as in DN-GSK3β cells (Fig. 7b). In uba1-dsRNA cells, the Ci155 levels are sensitive to CSN-mediated deneddylation because of insufficient activated Ub (Fig. 7c). In these cases, we envision that processive polyubiquitylation becomes more difficult because of constant neddylation–deneddylation cycling by the CSN, and can be aborted by the dissociation of Ci155 from weakly associated SCFSlimb-Ci155 or the lack of activated Ub. However, an insufficiency in Ci155 downregulation can be offset by constitutive neddylated SCFSlimb in CSN mutants, which ubiquitylates substrates processively on a single SCFSlimb-Ci155 encounter (Fig. 7d,e). This model explains the expression of CSN-dependent conditionally stable Ci155 in the low-to-intermediate Hh signalling regions, in which Ci155 may be released from the labile SCFSlimb-Ci155 complex before SCF re-neddylation.


CSN-mediated deneddylation differentially modulates Ci(155) proteolysis to promote Hedgehog signalling responses.

Wu JT, Lin WH, Chen WY, Huang YC, Tang CY, Ho MS, Pi H, Chien CT - Nat Commun (2011)

Models for SCFSlimb-mediated Ci155 proteolysis.Successful substrate polyubiquitylation requires collaboration of three components: adequate substrate–enzyme interaction time, persistent neddylated E3 ligase activity and high levels of activated ubiquitin supply. Green dot, ubiquitin; blue dot, Nedd8; and red dot, phosphate group. (a) For fully phosphorylated Ci155 that binds strongly to SCF in no-to-low Hh signalling regions, long substrate–enzyme binding duration allows complete polyubiquitylation, despite temporary inactivation of SCF ligase by CSN-mediated deneddylation. (b) In low-to-intermediate Hh signalling regions as well as in the presence of DN-GSK3β, partially phosphorylated Ci155 associates with SCF weakly. Dissociation of Ci155 from the SCFSlimb complex following CSN-mediated deneddylation of SCF disrupts processive ubiquitylation of Ci155. (c) Limited supply of activated ubiquitin could disrupt processive ubiquitylation of fully phosphorylated Ci155 in no-to-low Hh regions in the presence of CSN deneddylation. (d) CSN mutations allow the processivity of polyubiquitylation mediated by constantly neddylated SCF, which compensates for the short duration of interaction between SCFSlimb and Ci155 in low-to-intermediate Hh signalling regions or in the presence of DN-GSK3β. (e) In no-to-low Hh regions, limited supply of activated ubiquitin in uba1-dsRNA cells interrupts Ci155 polyubiquitylation, which is suppressed by constitutively neddylated SCF in CSN mutants.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3105314&req=5

f7: Models for SCFSlimb-mediated Ci155 proteolysis.Successful substrate polyubiquitylation requires collaboration of three components: adequate substrate–enzyme interaction time, persistent neddylated E3 ligase activity and high levels of activated ubiquitin supply. Green dot, ubiquitin; blue dot, Nedd8; and red dot, phosphate group. (a) For fully phosphorylated Ci155 that binds strongly to SCF in no-to-low Hh signalling regions, long substrate–enzyme binding duration allows complete polyubiquitylation, despite temporary inactivation of SCF ligase by CSN-mediated deneddylation. (b) In low-to-intermediate Hh signalling regions as well as in the presence of DN-GSK3β, partially phosphorylated Ci155 associates with SCF weakly. Dissociation of Ci155 from the SCFSlimb complex following CSN-mediated deneddylation of SCF disrupts processive ubiquitylation of Ci155. (c) Limited supply of activated ubiquitin could disrupt processive ubiquitylation of fully phosphorylated Ci155 in no-to-low Hh regions in the presence of CSN deneddylation. (d) CSN mutations allow the processivity of polyubiquitylation mediated by constantly neddylated SCF, which compensates for the short duration of interaction between SCFSlimb and Ci155 in low-to-intermediate Hh signalling regions or in the presence of DN-GSK3β. (e) In no-to-low Hh regions, limited supply of activated ubiquitin in uba1-dsRNA cells interrupts Ci155 polyubiquitylation, which is suppressed by constitutively neddylated SCF in CSN mutants.
Mentions: In this study, we show that CSN-mediated deneddylation of SCFSlimb is critical for the Ci155 stability in low-to-intermediate Hh regions in which partially phosphorylated Ci155 has a reduced affinity for the ubiquitylation machinery SCFSlimb. The effect of CSN deneddylation on SCFSlimb likely reduces ubiquitylation processivity, thus preserving 'conditionally stable Ci155' in these low-to-intermediate Hh regions. We propose a broader view for CRL substrate degradation in vivo, determined by three interdependent factors: substrate-enzyme affinity, deneddylation-regulated CRL activity and the supply of Uba1-activated Ub. We propose that steady-state Ci155 levels are low in the low Hh signalling regions because processive polyubiquitylation of the tightly associated SCFSlimb-Ci155 complex proceeds even with intermittent SCFSlimb inactivation by CSN-mediated deneddylation (Fig. 7a). In low-to-intermediate Hh regions, the steady-state Ci155 levels are sensitive to CSN-mediated deneddylation as a result of weakened SCFSlimb-Ci155 association, as in DN-GSK3β cells (Fig. 7b). In uba1-dsRNA cells, the Ci155 levels are sensitive to CSN-mediated deneddylation because of insufficient activated Ub (Fig. 7c). In these cases, we envision that processive polyubiquitylation becomes more difficult because of constant neddylation–deneddylation cycling by the CSN, and can be aborted by the dissociation of Ci155 from weakly associated SCFSlimb-Ci155 or the lack of activated Ub. However, an insufficiency in Ci155 downregulation can be offset by constitutive neddylated SCFSlimb in CSN mutants, which ubiquitylates substrates processively on a single SCFSlimb-Ci155 encounter (Fig. 7d,e). This model explains the expression of CSN-dependent conditionally stable Ci155 in the low-to-intermediate Hh signalling regions, in which Ci155 may be released from the labile SCFSlimb-Ci155 complex before SCF re-neddylation.

Bottom Line: Here, we show that in COP9 signalosome (CSN) mutants, in which deneddylation of SCF(Slimb) is inactivated, Ci is destabilized in low-to-intermediate Hh signalling cells.The status of Ci phosphorylation and the level of E1 ubiquitin-activating enzyme are tightly coupled to this CSN regulation.We propose that the affinity of substrate-E3 interaction, ligase activity and E1 activity are three major determinants for substrate ubiquitylation and thereby substrate degradation in vivo.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Medicine, College of Medicine, National Taiwan University, Taipei 100, Taiwan.

ABSTRACT
The Hedgehog (Hh) morphogen directs distinct cell responses according to its distinct signalling levels. Hh signalling stabilizes transcription factor cubitus interruptus (Ci) by prohibiting SCF(Slimb)-dependent ubiquitylation and proteolysis of Ci. How graded Hh signalling confers differential SCF(Slimb)-mediated Ci proteolysis in responding cells remains unclear. Here, we show that in COP9 signalosome (CSN) mutants, in which deneddylation of SCF(Slimb) is inactivated, Ci is destabilized in low-to-intermediate Hh signalling cells. As a consequence, expression of the low-threshold Hh target gene dpp is disrupted, highlighting the critical role of CSN deneddylation on low-to-intermediate Hh signalling response. The status of Ci phosphorylation and the level of E1 ubiquitin-activating enzyme are tightly coupled to this CSN regulation. We propose that the affinity of substrate-E3 interaction, ligase activity and E1 activity are three major determinants for substrate ubiquitylation and thereby substrate degradation in vivo.

Show MeSH
Related in: MedlinePlus